【摘要】 本文比较了前人提取致癌农杆菌(Agrobacterium tumefaciens)Ti质粒的方法,改变了溶菌的条件和蛋白质除去的程序,找到一种简易、快速、重复性高的提取Ti质粒的方法。本文的方法是将一份致癌农杆菌细胞同四份碱性SDS溶菌液(pH12.6)混合,这样溶菌液的pH为10.5,然后将此碱性溶菌液(pH10.5)置37℃保温30分钟。为了有效地提取Ti质粒又改变了去除蛋白质的程序,溶菌液首先用苯酚—氯仿抽提,随之加5M NaCl于溶菌液中,使NaCl最终浓度达0.6M,溶菌液于冰水中盐析2小时,悬浮的溶菌液经高速高心,大部分蛋白质和变性DNA被除去,DNA提取液由混浊变清。这种方法也可以用于提取其它菌株中的小分子质粒。更多还原

【Abstract】 A detailed procedure for rapid isolation of Ti plasmid from Agrobacterium tumefaciens was described. The pH 10.5 for the lysis of Agrobacterium cells is better than pH 9.5 and 12. The suspension of bactierial culture was mixed with four times volumes of the alkali sodium dodecyl sulface (pH 12.6) lysate. The alkali lysate (pH 10.5) was incubated at 37℃ for 30 minutes. For removing protein, the lysate was extracted with phenol-chloroform, followed by adding sufficient NaCl (5M NaCl) to bring the solution to 0.6M NaCl, then put the lysate on ice for two hours. Most of the proteins and the denatured chromosome DNA were thus removed and the tubid aqueous DNA solution turned to be clear. This technique is also proved to be useful in isolating small plasmid from a variety of other bucteria.更多还原